Hemophilia A is an inherited bleeding disorder caused by deficiency of Factor VIII, a blood coagulation protein that functions as a cofactor in the coagulation cascade. It is transmitted as a defect on the X chromosome and affects 1 male in 5000. Recent advances in biotechnology and protein engineering, together with cloning of the gene coding for Factor VIII, have made it feasible to manufacture recombinant human Factor VIII (rAHF). The recombinant preparation promises to be a source of unlimited supply, together with the freedom from the complications of transmission of blood-borne viruses. However, rAHF induces antibody type immune response or inhibitors. It has been shown that immune response for a therapeutic protein is caused by the existence of aggregates, frequent administration and existence of natural antibodies for specific epitope region of the protein. Furthermore, a moderately-short circulating half-time limits the duration of pharmacological effect. The overall goal of this proposal is to develop a lipid-based delivery vehicle for AHF to overcome the immune response and extend circulating halflife (Inhibitor Treatment for Hemophilia using Lipid Protein System, ITHELPS). The rationale to use lipidic protein complexes is three fold: (1) the epitope region of the protein binds to phospholipids, and thus by engineering a lipid-protein complex one could shield the epitope region; (2) the lipidic components can interact with intermediate structures in the unfolding/denaturation pathway, thus stabilizing protein structure during storage and reconstitution, thereby avoiding protein aggregate formation, and (3) the lipid-protein complexes, analogous to liposomal particles, may increase the circulation half-life of the rAHF, thus reducing the frequency of administration. In support of these hypotheses, preliminary studies were carried out to develop rationally a lipidic rAHF. The liposomal vesicles stabilized the protein against aggregation and a prototype formulation with lipidic rAHF was achieved. We propose to characterize the molecular topology, pharmacokinetic behavior and immune response of this novel, rationally developed lipidic rAHF. Further, we propose to investigate liposomal clearance mechanisms, including endocytosis and its role in altering the circulation time and immune response of lipidic rAHF. We also propose to study the epitope specificity of rAHF antibodies and the mechanism of immune response.